摘 要

本篇論文的研究目的是了解調控及功能型蛋白的特性及其應用在植物逆境上的應用。本論文主要分為三個章節。第一章主要探討一個胚胎發育後期大量累積的蛋白質(HVA1) 在逆境耐受性中的作用；第二章是研究水稻轉錄因子MYBS2在糖訊息傳導中所扮演的角色；第三章是探討水稻轉錄因子MYBS1的兩個阿拉伯芥同源基因在糖訊息傳導中所扮演的角色。
調節根系結構對於植物在惡劣環境環境下維持生長是至關重要的。一個逆境誘導型啟動子被用來調控HVA1基因的表現量。第一章中，發現HVA1在受逆境或是ABA誘導後會大量累積在根冠分生組織（RAM）及側根原基（LRP），進而促進根系發育。即使在ABA或逆境抑制狀況下，HVA1還是可以促進側根的起始，延長及生長，以及初級根系的向下延長，而這是透過在側根原基和根冠分生組織中增加不對稱的植物生長素（auxin）累積所致。在本實驗中成功應用一個誘導型啟動子來調控HVA1的時空表現，不僅讓水稻可以增進根系發育以及在多種逆境下具有抗性，而且不會造成水稻產量損失。
自營植物已經進化出獨特的機制來維持糖的平衡。有糖、缺糖相互調控基因表現的開與關是維持糖恆定的主要機制之一，但其細節卻尚不明。α-澱粉水酵素（αAmy）是將澱粉水解為植物生長所需醣類的關鍵酵素，它會被缺糖誘導與有糖抑制。在本章中，αAmy基因表現量的開與關主要是受到兩個MYB轉錄因子競爭同一個啟動子的鍵結位置所調控。在缺糖下，MYBS1促進Amy的表現，而有糖下MYBS2抑制它。缺糖促進MYBS1進核與MYBS2出核，反之有糖則產生相反的效果。MYBS2蛋白質上不同位置的絲氨酸殘基(serine)的磷酸化是調控MYBS2在不同糖環境下的進、出核以及維持其與14-3-3蛋白質的交互作用主要因子。此外，脫水、熱與滲透逆境皆會抑制MYBS2的表現，進而誘導Amy3 表現。最重要的是，大量表現Amy3與抑制MYBS2皆可以增進水稻的生長、逆境的耐受性和單株總粒重。
水稻轉錄因子OsMYBS1參與糖和激素所調節的Amy基因表現量。第三章中，研究了水稻轉錄因子MYBS1的兩個阿拉伯芥同源基因MYBS1與MYBS2在糖訊息傳導中的作用。在種子發芽與幼苗生長過程中，atmybs1突變株對糖與ABA非常敏感，而atmybs2突變株則對葡萄糖與ABA較不敏感。此外，atmybs1突變株會誘導葡萄糖反應性基因（如HXK1，CAB1，APL3和CHS）的表現量，而atmybs2突變株則抑制這些基因的表現量。在糖處理後，atmybs1突變株中的三個ABA生合成基因（ABA1，NCED9與AAO3）以及三個ABA訊息傳導基因（ABI3，ABI4與ABI5）的表現量提升，而這些基因的表現量在atmybs2突變株中則降低。這些結果顯示在ABA依賴性的糖抑制種子發芽和幼苗發育過程中，AtMYBS1具有負向調節的作用，而AtMYBS2則具有正向調節的作用。
這些研究中對於相關基因的瞭解將有助於培育具有逆境抗性的品種以其達到永續農業的目的。

Abstract

The aim of this study is to characterize the regulatory and functional proteins that confer the stress tolerances in plants. The dissertation includes three chapters. The first chapter examines the roles of a late embryogenesis abundant protein (HVA1) in stress tolerance. The second chapter discribes the roles of MYBS2 transcription factor in sugar signaling and stress tolerance in rice. The final chapter demonstrates the roles of two MYB transcription factors in Arabidopsis.
Regulation of root architecture is essential for maintaining plant growth under adverse environments. Stress/ABA inducible HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. HVA1 promotes lateral root (LR) initiation, elongation and emergence and primary root (PR) elongation via an auxin-dependent process, particularly by intensifying asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress-suppressive conditions. We demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty.
Autotrophic plants have evolved distinctive mechanisms for maintaining a range of sugars homeostatic states. The on/off switch of reversible gene expression by sugar starvation/provision represents one of the major mechanisms by which sugar levels are maintained, but the details remain unclear. -Amylase (Amy) is the key enzyme for hydrolyzing starch into sugars for plant growth. It is induced by sugar starvation and repressed by sugar provision. In the second chapter, the on/off switch of Amy expression was found to regulate by two MYB transcription factors competing for the same promoter element. MYBS1 promotes Amy expression under sugar starvation, whereas MYBS2 represses it. Sugar starvation promotes nuclear import of MYBS1 and nuclear export of MYBS2, whereas sugar provision has the opposite effects. Phosphorylation of MYBS2 at distinct serine residues plays important roles in regulating its sugar-dependent nucleocytoplasmic shuttling and maintenance in cytoplasm by 14-3-3 proteins. Moreover, dehydration, heat and osmotic stress repress MYBS2 expression, thereby inducing Amy3. Importantly, activation of Amy3 and suppression of MYBS2 enhances plant growth, stress tolerance and total grain weight per plant in rice.
OsMYBS1 is involved in sugar- and hormone-regulated α-amylase gene expression in rice. In the third chapter, the roles of AtMYBS1 and AtMYBS2, which are OsMYBS1 homologs, in sugar signaling in Arabidopsis were investigated. Germination and seedling growth of atmybs1 mutant were hypersensitive, whereas those of atmybs2 were hyposensitive, to glucose and ABA. Furthermore, the expression of glucose-responsive genes, such as HXK1, CAB1, APL3, and CHS, were up-regulated in the atmybs1 mutant but down-regulated in the mybs2 mutant. Moreover, the mRNA levels of three ABA biosynthesis genes, ABA1, NCED9, and AAO3, and three ABA signaling genes, ABI3, ABI4, and ABI5, were increased upon glucose treatment in atmybs1, but decreased in mybs2, seedlings. These results suggest that AtMYBS1 plays a negative, while AtMYBS2 plays a positive, role in the ABA-dependent sugar repression of germination and seeding development.
The knowledge, gene, and findings obtained from these studies will help in developing tolerance crop varieties for sustainable agriculture.

Table of contents
摘 要 i
Abstract iii
誌 謝 v
Table of contents vii
List of supplementary figures x
List of tables xi
Chapter I -General introduction 1
References 13
Chapter II- A Late Embryogenesis Abundant Protein HVA1 Regulated by an Inducible Promoter Enhances Auxin-dependent Root Growth and Tolerance to Abiotic Stresses in Rice without Yield Penalty 25
Abstract 25
2.1 Introduction 26
2.2 Material and Methods 29
2.3 Results 32
2.4 Discussion 36
References 40
Chapter III- Sugar starvation-regulated interactions between MYBS2 and 14-3-3 proteins enhances plant growth, stress tolerance and grain yield in rice 62
Abstract 62
3.1 Introduction 63
3.2 Material and Methods 66
3.3 Results 72
3.4 Discussion 83
References 91
Chapter IV- Two MYB-related transcription factors play opposite roles in sugar signaling in Arabidopsis 125
Abstract 125
4.1 Introduction 127
4.2 Material and Methods 129
4.3 Results 133
4.5 Discussion 139
References 143
Chapter V-Future perspectives 164
References 170

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